Monitoring signals of microbial invasion is universally the first step in mounting an immune response. Yet we lack a full understanding of the systems and mechanisms that influence immunity. My long term goal seeks to understand the processes that drive the activation of intracellular sensor proteins that recognize microbial molecules. These intracellular receptors belong to the nucleotide-binding domain (NBD) leucine-rich repeat (LRR) protein family (generically termed NLRs), and are critical components of innate immune systems. Mutations in this class of genes have been implicated in human disease. Considering their importance across kingdoms for human health and crop productivity our current understanding of NLR protein function at the molecular level is rudimentary. Knowledge transfer between model organisms and human biology has accelerated prominent discoveries and thus understanding NLR function and activation will require a concerted interdisciplinary approach. I will use a prototypical plant NLR, Resistance to Pseudomonas maculicola 1 (RPM1) as a functional model for NLR activation. RPM1 indirectly recognizes two unrelated pathogenic bacterial type III effector proteins (AvrB and AvrRpm1) via effector-induced phosphorylation of the RPM1- interacting 4 (RIN4) protein. This recognition event activates a complex output response, resulting in programmed cell death at the infection site and restriction of pathogen colonization. Recent published and unpublished data from our laboratory suggests phosphorylated RIN4 associates with RPM1 with a greater affinity than does unphosphorylated RIN4. I hypothesize effector-induced phosphorylation of RIN4 enhances electrostatic interactions between the N-terminal coiled-coil (CC) domain of RPM1 and RIN4 entailing a stable interaction and leading to a RPM1 conformational change that relieves auto-inhibition imposed by the RPM1 CC and LLR domains. This enables nucleotide exchange/hydrolysis and subsequent downstream immune signaling. My short term goals are to test my proposed model and examine the physical attributes of RPM1 activation using a mixture of genetic and biochemical approaches highlighted in this proposal. The proposed research will shed light on a poorly understood NLR activation process.